This invention relates generally to the field of detection of bacteria, and more specifically to the diagnosis of actinomycetes infection, particularly to those caused by mycobacteria. The invention also relates generally to the field of diagnosis of bacterial infection, and more specifically to the diagnosis of diseases associated with actinomycetes infection such as tuberculosis.
Human tuberculosis is caused primarily by the bacterium Mycobacterium tuberculosis. Although other species of mycobacteria can cause human disease, M. tuberculosis is by far the most important cause of morbidity and mortality among the mycobacterial genus. It is estimated that 8.8 million new cases of tuberculosis occurred in 1995 and these numbers are projected to continue to increase.
The laboratory diagnosis of tuberculosis has always been complicated by the slow growth of M. tuberculosis in culture. Visible growth on solid culture media can require up to 8 weeks of incubation. Presumptive diagnoses are based on finding mycobacteria by microscopic examination of a diagnostic specimen often an expectorated sputum. These smears are either stained by acid fast stains like the Ziehl-Neelsen stain or by auramine-rhodamine staining followed by fluorescence microscopy. Smears must contain 5xc3x97103 to 105 bacteria per ml for detection and are nonspecific since they recognize all species of mycobacteria (Nolte, F. S., and Metchock, B., 1995, xe2x80x9cMycobacterium.xe2x80x9d In: Manual of Clinical Microbiology, 6th Edition., Murray, P. R. (ed.), ASM Press, Washington, D.C.) Definitive diagnosis is dependent on the isolation and identification of M. tuberculosis in culture of a diagnostic specimen, usually a sputum obtained from a patient with a productive cough (Nolte and Metchock, 1995, supra). Although the use of liquid cultures with radiometric detection methods and the identification of cultivated bacteria by nucleic acid probes have shortened the time to isolate and identify M. tuberculosis, definitive diagnosis still usually requires 2-3 weeks (Raviglione, M. D., and O""Brien, R. J., 1998, xe2x80x9cTuberculosis,xe2x80x9d In: Harrison""s Principles of Internal Medicines, 14th Edition, Fauci et al., eds., McGraw-Hill, N.Y.).
The conventional technique for detecting tuberculosis is by microscopic identification of the bacteria in patient specimens treated with special stains combined with cultivation on specific bacteriologic media. Detection by the staining techniques is nonspecific and relatively insensitive, and cultivation is time-consuming and expensive because Mycobacterium tuberculosis grows very slowly. Detection of M. tuberculosis by nucleic acid amplification is an adjunctive approach. Because of the duration of time required to establish a definitive diagnosis, many adjunctive laboratory diagnostic tests have been investigated. Serologic detection of antibody to M. tuberculosis has had very low predictive values (Daniel, T. M., and Debanne, S. M., 1987, Am. Rev. Respir. Dis. 158:678). Serologic detection of tuberculostearic acid, another unique component of actinomycetes, by gas chromatography and mass spectroscopy with selective ion monitoring has been too costly to be implemented outside of research laboratories (Brooks et al., 1990, J. Clin. Microbiol. 28:98; Nolte and Metchock, 1995, supra).
Mycothiol (MSH) is a recently discovered novel cysteine derivative produced only by actinomycetes. Among the actinomycetes, mycobacteria produce mycothiol in the greatest amounts. Mycobacteria are the main group of actinomycetes that infect humans. Among these infections are tuberculosis (TB), as well as other mycobacterial infections. Other genera of actinomycetes (corynebacteria, including the causative agent of diphtheria, and Nocardia species, which cause pulmonary nocardiosis) make MSH, but they are minor causes of human morbidity and mortality compared to tuberculosis (Newton et al., 1996, J. Bacteriol., 178).
The available information on mycothiol is quite limited and very recent. The structure for mycothiol, 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-xcex1-D-glucopyranoside (MSH) (FIG. 1), was first reported by Sakuda et al. (1994, Biosci. Biotech. Biochem., 58:1347) who isolated it as its disulfide from a Streptomyces species. It was later isolated as the free thiol from Mycobacterium bovis (Spies, H. S. C., and Steenkamp, D. J., 1994, Eur. J. Biochem., 224:203) and Streptomyces clavuligerus (Newton et al., 1995, Eur. J. Biochem., 230:821). The name xe2x80x9cmycothiolxe2x80x9d (Spies and Steenkamp, 1994, supra) and the abbreviation MSH (Newton et al., 1995, supra) have been proposed for this unusual thiol.
The actinomycetes do not produce glutathione (GSH), a thiol antioxidant found in many prokaryotes and eukaryotes (Fahey, R. C., and Sundquist, A. R., 1991, Adv. Enzymol. Relat. Areas Mol. Biol. 64:1). Instead, most actinomycetes (but not other prokaryotes or eukaryotes) produce MSH at millimolar levels (Newton et al., 1996, J. Bacteriol., 178:1990-1995). MSH has superior antioxidant properties to GSH (Newton et at., 1995, supra) and thus may serve both as a stable intracellular storage form of cysteine and as the essential cofactor for oxidative stress-response and detoxification enzymes in a manner analogous to that of GSH in GSH-producing organisms. Though little is currently known about the biochemistry of MSH, it has recently been reported that MSH is the cofactor for an NAD/xe2x80x9ccofactorxe2x80x9d-dependent formaldehyde dehydrogenase found in actinomycetes, where it serves in a detoxification role analogous to that of GSH in the NAD/GSH-dependent formaldehyde dehydrogenase of cukaryotes and Gram-negative bacteria (Misset-Smits, M., et al., 1997, FEBS Lett. 409:221-222). It is likely that further examples of MSH-dependent protective enzymes will be found in the future. Thus enzymes involved in the metabolism of MSH may represent targets for new drugs directed against tuberculosis and other mycobacterial infections (Newton et al., 1996, supra).
Among the actinomycetes, mycobacteria produce the highest levels of mycothiol, xcx9c5 million molecules per cell, and as few other actinomycetes besides mycobacteria are human pathogens, the present inventors have thus proposed that detection of MSH is a possible way to screen for tuberculosis and other mycobacterial infections. The development of sensitive and specific methods for the detection of MSH is essential for both research in the elaboration of mycothiol metabolism and for use in clinical diagnosis of mycobacterial infection. MSH analysis has previously relied on derivatization with thiol-specific fluorescent labeling reagents followed by HPLC analysis (Spies and Steenkamp, 1994, supra; Newton et al., 1993, J. Bacteriol., 175:2734), but this methodology is expensive, time-consuming, lacks the sensitivity needed to be clinically useful in the diagnosis of mycobacterial infection, and lacks the versatility needed for a variety of applications such as the screening for mycothiol production by bacterial colonies.
The present invention is based on the discovery of methods for detecting mycothiol, a recently described novel cysteine derivative produced at millimolar intracellular levels by actinomycetes, including the pathogenic mycobacteria.
A method of detecting a member of the taxa actinomycetes is provided. The method includes incubating a reagent that detects mycothiol or a precursor thereof with a sample for a time sufficient for said reagent to react with mycothiol or precursor thereof, and detecting the reaction of the reagent with mycothiol or a precursor thereof. In this method detection of a reaction is indicative of the presence of a member of the taxa actinomycetes.
An antibody is provided which binds to mycothiol or a mycothiol precursor. The antibody may be a monoclonal or a ployclonal antibody.
A method is provided for diagnosis of a subject having or at risk of having an Actinomycetes-associated disorder. The method includes contacting a sample from the subject having or at risk of having an actinomycetes-associated disorder with a reagent that detects mycothiol or precursor thereof for a period of time sufficient for said reagent to react, and detecting the reaction of the reagent with mycothiol or precursor thereof. The reaction of the reagent that detects mycothiol or precursor thereof to the sample is compared to a control sample.
A method is provided for identifying a sample with altered production of mycothiol or a precursor thereof, including contacting a test sample with a reagent that detects mycothiol or precursor thereof for a period of time sufficient for the reagent to react, and detecting the reaction of the reagent with mycothiol or precursor thereof. The reagent that detects mycothiol or precursor thereof to the test sample is compared with a control sample; a difference in the amount of reaction with the test sample as compared to the control sample is indicative of an alteration in the production of mycothiol or precursor thereof.
A method is provided for detecting mycothiol or precursor thereof in a bacterial colony, including contacting a membrane to a bacteria plated on a bacterial culture plate for a time sufficient to allow the bacteria to adhere to the membrane and lysing the bacteria. The method also includes contacting the membrane with a reagent to detect mycothiol or precursor thereof, and detecting said the reaction of reagent with mycothiol or precursor thereof.
A method is further provided for detecting mycothiol or precursor thereof including biotinylating mycothiol or precursor thereof to form biotinylated mycothiol or biotinylated mycothiol precursor, and contacting the biotinylated mycothiol or biotinylated mycothiol precursor to an antibody with binds mycothiol or precursor thereof to form a complex. The method further includes detecting the presence of said complex with a detection reagent.
Kits are also disclosed which are useful for detecting the presence of mycothiol or precursor thereof in a sample.
A method is also provided for detecting a mycothiol or a precursor thereof, including partially purifying mycothiol or a precursor thereof, reacting the precusor of mycothiol with a reagent for fluroescent amine labeling to form a reaction product; and detecting the presence of the reaction product. Detecting may be at a pH range from about 7.5 to 9, and more likely from about 8.0 to 8.6.